We propose to develop an ultrasound technology for contrast-enhanced imaging of infused platelets as they interact to form arterial thrombi. The ability to detect thrombosis at a clinically silent, pre-occlusive stage can potentially identify patients before the thrombi become occlusive and cause heart attacks, strokes, or gangrenous extremities. This technology will also guide the development, using relevant animal models, of new interventional strategies for minimizing thrombotic complications in atherosclerosis. There are currently no existing clinical imaging methods to directly contrast platelets or to detect pre-occlusive, clinically silent thrombosis. Superparamagnetic iron oxides (SPIOs) have recently emerged as a new contrast agent for ultrasound that offer dark-field imaging via lock-in detection of induced magnetomotion of echogenic materials (cells or blood clots) with which SPIOs are mechanically coupled. We hypothesize that rehydratable, lyophilized (RL) platelets carrying a payload of SPIOs can provide functional contrast to arterial thrombosis via magnetomotive ultrasound (MMUS), while enabling long-term storage capability. Our research strategy employs a perfusion chamber for real-time imaging of ex vivo porcine arteries as a flexible platform to characterize this new imaging technology. As a model of vascular endothelial reactivity, we will first de-endothelialize arteries as an injury model, or add TNF cytokine to induce inflammation of ex vivo porcine arteries. Then, as a model of thrombosis complicating atherosclerosis, we will study arteries from hypercholesterolemic pigs that have atherosclerotic plaques. Our first specific aim is to determine the minimum circulating concentration of SPIO-RL platelets needed to provide MMUS contrast during thrombus formation, which will provide baseline data to inform clinical feasibility. As part of this aim we will study the effects of flowrate on thrombus formation, and thrombus size on MMUS contrast, while optimizing imaging parameters. An exploratory component of this aim is proposed to investigate whether the stiffness of the thrombus can be extracted via the amplitude and frequency-dependence of the MMUS signals, which may be of relevance for identifying disorders such as hyperfibrinogenemia. Our second specific aim is to determine the fate of SPIOs after SPIO-RL platelets have been incorporated into a thrombus, in order to predict pharmacokinetics and to inform future strategies such as therapeutic payloads. The active and essential role that platelets play in thrombus formation is a desirable platform for functional imaging, which is expected to provide improved sensitivity over mono-functionally targeted agents such as antibody-conjugated SPIOs. This proposed work will validate the efficacy of SPIO-RL platelets for thrombosis detection in hemodynamic conditions, and may lead to significant improvement in the ability to reveal atherosclerotic plaque hemorrhage or preclinical, nonocclusive thrombi, justifying clinical interventions to mitigate occlusive thrombosis.